CN112147240A - Extraction and detection method of aflatoxin in spina date seeds - Google Patents
Extraction and detection method of aflatoxin in spina date seeds Download PDFInfo
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Abstract
The invention discloses an extraction and detection method of aflatoxin in spina date seeds, which comprises the steps of crushing the spina date seeds, sieving the crushed spina date seeds by a second sieve to obtain spina date seed powder, placing the spina date seed powder in a centrifuge tube with a plug, adding methanol or acetonitrile, carrying out vortex oscillation, and then carrying out ultrasonic treatment for 5-30 min at an ultrasonic power of 100-500W; adding anhydrous sodium sulfate or anhydrous magnesium sulfate or sodium chloride, mixing by vortex, and centrifuging at rotating speed to obtain supernatant; taking the supernatant into a centrifugal tube, adding octadecylsilane chemically bonded silica or N-propylethylenediamine for purification, carrying out vortex mixing, centrifuging at a rotating speed, and filtering by an organic phase filter membrane to obtain a solution to be detected; and filtering the standard aflatoxin use solution by using an organic phase filter membrane, and detecting the solution to be detected by using a liquid chromatography-tandem mass spectrometer. The method is simple and rapid to operate, reduces the interference of the sample matrix on the target object, reduces the matrix effect, and has the advantages of high sensitivity, less pollution, lower detection cost and easy popularization.
Description
Technical Field
The invention relates to the technical field of extraction and detection of traditional Chinese medicines, and particularly relates to an extraction and detection method of aflatoxin in spina date seeds.
Background
The aflatoxin is a secondary metabolite of a toxigenic strain of aspergillus flavus and aspergillus parasiticus, is a derivative of dihydrofurocoumarin structurally, has far higher toxicity than cyanide, iodide and organic pesticide, is a fungaltoxin discovered to be the strongest in toxicity so far, is commonly found in plant-derived agricultural and sideline products such as peanuts, grains, Chinese medicinal materials and the like, has harmfulness in destroying liver tissues of people and animals, and can cause liver cancer and even death when serious.
The spina date seed has the effects of tonifying liver, calming heart, arresting sweating and promoting fluid production, is used for treating dysphoria, insomnia, palpitation and dreaminess, body deficiency and hyperhidrosis, body fluid deficiency and thirst and the like, is listed as the top grade in the compendium of materia medica, but the spina date seed is easy to mildew, so that various mycotoxins harmful to human bodies are generated, wherein the aflatoxin is the most harmful, currently, the determination of the aflatoxin in China is mainly focused on grains, peanuts and products thereof, milk and products thereof and the like, and the detection method for the aflatoxin in the traditional Chinese medicinal materials is relatively laggard.
Common detection methods for aflatoxin include thin layer chromatography, high performance liquid chromatography, enzyme-linked immunosorbent assay and the like. The thin-layer chromatography is the earliest separation and analysis technology of aflatoxin and the widest application, but the early-stage treatment of a sample is complicated, and the extracting solution has more impurities, so that the requirements of modern analysis cannot be met; the high performance liquid chromatography requires the derivation of a sample, the operation is complex and the analysis time is long; the complex matrix of the enzyme-linked immunosorbent assay is easy to cause interference and false positive; at present, the method for detecting aflatoxin in 'Chinese pharmacopoeia' 2015 edition adopts an immunoaffinity column to purify a sample, but the method has complex operation and higher cost.
Disclosure of Invention
In view of the above, the invention is expected to provide a method for extracting and detecting aflatoxin in spina date seeds, which is simple and rapid to operate, reduces the interference of a sample matrix on a target object, reduces a matrix effect, and is high in sensitivity, less in pollution, lower in detection cost and easy to popularize.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
the invention provides a method for extracting and detecting aflatoxin in spina date seeds, which comprises the following steps:
a) crushing the spina date seeds, sieving the crushed spina date seeds by a second sieve to obtain spina date seed powder, placing the spina date seed powder into a centrifuge tube with a plug, and adding methanol or acetonitrile into the centrifuge tube at a material-to-liquid ratio of 1: 1-1: 100; carrying out vortex oscillation for 1-10 min at the rotating speed of 1000-3000 r/min, and then carrying out ultrasonic treatment for 5-30 min at the ultrasonic power of 100-500W; adding 0.1-20 g of anhydrous sodium sulfate or anhydrous magnesium sulfate or sodium chloride, mixing for 1-10 min in a vortex mode at the rotating speed of 1000-3000 r/min, centrifuging for 8-16 min at the rotating speed of 1500-4500 r/min, and taking supernatant;
b) taking supernatant fluid into a centrifuge tube, adding octadecylsilane chemically bonded silica or N-propyl ethylenediamine for purification, carrying out vortex mixing at the rotating speed of 1000-3000 r/min for 1-10 min, centrifuging at the rotating speed of 1500-4500 r/min for 3-10 min, and filtering through an organic phase filter membrane with the pore diameter of 0.1-0.6 mu m to obtain a solution to be detected;
c) filtering standard aflatoxin use solution by an organic phase filter membrane with the aperture of 0.1-0.6 μm, and detecting the solution to be detected by adopting a liquid chromatography-tandem mass spectrometer, wherein a chromatographic column is Unitry C18, a mobile phase A is ammonium formate or ammonium acetate or formic acid, a mobile phase B is methanol, the flow rate is 0.2-0.5 mL/min, the column temperature is 25-40 ℃, the sample injection volume is 1-10 μ L, the ionization mode is electrospray positive ion ionization, and the detection mode is multi-ion reaction monitoring.
Furthermore, the aperture of the organic phase filter membrane is 0.2-0.5 μm.
Furthermore, the chromatographic column filler is alkylsilane bonded silica, the length of the chromatographic column is 100 mm-250 mm, the inner diameter is 2.1 μm-4.6 μm, and the particle size is 1.7 μm-5 μm.
Further, after vortex oscillation is carried out for 3min at the rotation speed of 4000r/min in the step a), ultrasonic treatment is carried out for 20min at the ultrasonic power of 500W, vortex mixing is carried out for 3min at the rotation speed of 4000r/min, and then centrifugation is carried out for 5min at the rotation speed of 4000 r/min.
Further, N-propyl ethylenediamine is added in the step b) for purification, the mixture is subjected to vortex mixing for 3min at the rotating speed of 4000r/min, and then the mixture is centrifuged for 3min at the rotating speed of 4000r/min, and then the mixture is filtered by an organic phase filter membrane with the aperture of 0.22 mu m.
Further, the step of measuring the aflatoxin standard use solution in the step c) comprises the following steps: taking aflatoxin mixed reference solution with known concentration, diluting with methanol or ethanol or acetonitrile solution with volume fraction of 50-90%, fixing volume, storing at 2-8 ℃ for 1-3 months, and preparing into standard aflatoxin use solution with different concentrations by using methanol with volume fraction of 50-90%.
Further, the volume fraction of the methanol is 70-90%.
The invention has the following beneficial effects: the method is simple and rapid to operate, reduces the interference of the sample matrix on the target object, reduces the matrix effect, and has the advantages of high sensitivity, less pollution, lower detection cost and easy popularization.
Drawings
FIG. 1 is a schematic flow chart of the method for extracting and detecting aflatoxin in spina date seeds;
FIG. 2 is a chromatogram of standard liquid aflatoxin B1 in example 1 of the present invention;
FIG. 3 is a chromatogram of standard liquid aflatoxin B2 in example 1 of the present invention;
FIG. 4 is a chromatogram of standard liquid aflatoxin G1 in example 1 of the present invention;
FIG. 5 is a chromatogram of standard liquid aflatoxin G2 in example 1 of the present invention;
FIG. 6 is a graph showing the standard curve of aflatoxin B1 in example 1 of the present invention;
FIG. 7 is a graph showing the standard curve of aflatoxin B2 in example 1 of the present invention;
FIG. 8 is a graph showing the standard curve of aflatoxin G1 in example 1 of the present invention;
FIG. 9 is a graph showing the standard curve of aflatoxin G2 in example 1 of the present invention.
Detailed Description
So that the manner in which the features and aspects of the invention can be understood in detail, a more particular description of the invention, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings.
Fig. 1 is a schematic flow chart of the method for extracting and detecting aflatoxin in spina date seeds, and the method specifically comprises the following steps:
step 101: crushing the spina date seeds, sieving the crushed spina date seeds by a second sieve to obtain spina date seed powder, placing the spina date seed powder into a centrifuge tube with a plug, and adding methanol or acetonitrile into the centrifuge tube at a material-to-liquid ratio of 1: 1-1: 100; carrying out vortex oscillation for 1-10 min at the rotating speed of 1000-3000 r/min, and then carrying out ultrasonic treatment for 5-30 min at the ultrasonic power of 100-500W; adding 0.1-20 g of anhydrous sodium sulfate or anhydrous magnesium sulfate or sodium chloride, mixing for 1-10 min in a vortex mode at the rotating speed of 1000-3000 r/min, centrifuging for 8-16 min at the rotating speed of 1500-4500 r/min, and taking supernatant;
preferably, the spina date seed powder is placed in a 50mL centrifuge tube with a plug, acetonitrile is added, the material-liquid ratio is 1:10, the mixture is subjected to vortex oscillation for 3min at the rotating speed of 4000r/min, ultrasonic treatment is performed for 20min at the ultrasonic power of 500W, 3-10 g of anhydrous sodium sulfate is added, vortex mixing is performed for 3min at the rotating speed of 4000r/min, then the mixture is centrifuged for 5min at the rotating speed of 4000r/min, and a supernatant is obtained;
step 102: taking supernatant fluid into a centrifuge tube, adding octadecylsilane chemically bonded silica or N-propyl ethylenediamine for purification, carrying out vortex mixing at the rotating speed of 1000-3000 r/min for 1-10 min, centrifuging at the rotating speed of 1500-4500 r/min for 3-10 min, and filtering through an organic phase filter membrane with the pore diameter of 0.1-0.6 mu m to obtain a solution to be detected;
here, the pore diameter of the organic phase filtration membrane is preferably 0.2 μm to 0.5. mu.m;
preferably, 1-5 mL of supernatant is added into a 5mL centrifuge tube, 100-500 mg of N-propyl ethylenediamine is added for purification, the mixture is subjected to vortex mixing at the rotating speed of 4000r/min for 3min, and then is subjected to centrifugation at the rotating speed of 4000r/min for 3min, and then is filtered by an organic phase filter membrane with the aperture of 0.22 mu m to obtain a solution to be detected;
step 103: filtering standard aflatoxin use solution by an organic phase filter membrane with the aperture of 0.1-0.6 microns, and detecting the solution to be detected by adopting a liquid chromatography-tandem mass spectrometer, wherein a chromatographic column is Unitry C18, a mobile phase A is ammonium formate or ammonium acetate or formic acid, a mobile phase B is methanol, the flow rate is 0.2-0.5 mL/min, the column temperature is 25-40 ℃, the sample injection volume is 1-10 muL, the ionization mode is electrospray positive ion ionization, and the detection mode is multi-ion reaction monitoring;
here, the aflatoxin standard use solution measuring step includes: taking aflatoxin mixed reference solution with known concentration, diluting with methanol or ethanol or acetonitrile solution with volume fraction of 50-90%, fixing the volume, and storing at 2-8 ℃ for 1-3 months, wherein when in use, methanol with volume fraction of 50-90% is used for preparing standard aflatoxin use solution with different concentrations;
here, the pore diameter of the organic phase filtration membrane is preferably 0.2 μm to 0.5. mu.m;
here, the methanol volume fraction is preferably 70% to 90%;
the chromatographic column packing is alkylsilane bonded silica, the length of the chromatographic column is 100 mm-250 mm, the inner diameter is 2.1 μm-4.6 μm, and the particle size is 1.7 μm-5 μm.
In the invention, the calculation method of the aflatoxin content comprises the following steps: and (3) drawing a standard curve by taking the mass concentration of the aflatoxin as an abscissa and the corresponding peak area as an ordinate, and calculating the content of each aflatoxin in the sample according to the standard curve of each aflatoxin and the peak area in the sample:
X＝(A×V×1000)/m
wherein: x represents the content of each aflatoxin in the sample, and the unit is mu g/kg;
a, the corresponding concentration of a sample in a standard curve according to an external standard method is in the unit of mu g/mL;
v, the volume of the extracting solution in the sample extraction process is mL;
m is the sample size in g.
Further details will be given below by way of specific examples.
Examples
Step 1: crushing a spina date seed sample, sieving the crushed spina date seed sample by a No. two sieve, weighing 5g of spina date seed powder, placing the weighed spina date seed powder into a 50mL centrifuge tube with a plug, adding 20mL acetonitrile, carrying out vortex oscillation for 3min at the rotating speed of 4000r/min, and then carrying out ultrasonic treatment for 20min at the ultrasonic power of 500W; adding 5g anhydrous sodium sulfate for dehydration, mixing for 3min at the rotation speed of 4000r/min in a vortex mode, centrifuging for 5min at the rotation speed of 4000r/min, and taking supernatant;
step 2: taking 2mL of supernatant into a 5mL centrifuge tube, adding 200mg of N-propyl ethylenediamine adsorbent for purification, carrying out vortex mixing at the rotating speed of 4000r/min for 3min, centrifuging at the rotating speed of 4000r/min for 3min, and filtering through an organic phase filter membrane with the pore size of 0.22 mu m to obtain a solution to be detected;
and step 3: taking 1mL of aflatoxin mixed control solution (labeled concentrations of aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2 are respectively 500 mug/L, 150 mug/L, 500 mug/L and 150 mug/L), diluting with 70% methanol, and diluting to 10mL to obtain intermediate standard solution (labeled concentrations of aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2 are respectively 50 mug/L, 15 mug/L, 50 mug/L and 15 mug/L), storing for three months at 2-8 ℃, and preparing a series of standard reference use solutions containing aflatoxin B2 and G2 with the concentration of 0.04 ng/mL-3 ng/mL and aflatoxin B1 and G1 with the concentration of 0.12 ng/mL-10 ng/mL by using methanol when in use;
and 4, step 4: filtering standard aflatoxin use solution with an organic phase filter membrane with the pore diameter of 0.22 mu m, and detecting the solution to be detected by adopting a liquid chromatography-tandem mass spectrometer to obtain chromatograms of aflatoxins B1, B2, G1 and G2 in figures 2-5, wherein the specific conditions are as follows:
chromatographic conditions are as follows: a chromatographic column: unitry C18(100 mm. times.3 mm,3 μm);
mobile phase: taking 10mmol/L ammonium acetate solution as a mobile phase A and methanol as a mobile phase B; gradient elution was performed as specified in table 1 below.
TABLE 1 gradient elution procedure
Flow rate: 0.3 mL/min;
column temperature: 25-40 ℃;
sample introduction amount: 5 mu L of the solution;
mass spectrum conditions: ionization mode, electrospray positive ion ionization;
detection mode: multiple Reaction Monitoring (MRM); spraying pressure: 7.0 Bar; heating air flow: 1000L/Hr; flow rate of drying gas: 1000L/Hr; temperature of the drying gas: 500 ℃; capillary voltage: 3.1 kV; taper hole voltage: 68V; flow rate of the taper hole: 150L/Hr. The ion pairs and collision voltage (CE) for each compound were monitored as shown in table 2 below.
TABLE 2 Mass spectrometric parameters of the four aflatoxins
Linear regression is carried out by taking the mass concentration x of the aflatoxin as an abscissa and the corresponding peak area y as an ordinate, standard curves of the aflatoxins B1, B2, G1 and G2 are shown in figures 6-9, a linear equation, a linear range, correlation coefficients and quantitative limit results are shown in table 3, and results show that the linear relation of the four aflatoxins is good, r is a linear relation of the four aflatoxins2Not lower than 0.9901.
TABLE 3 Linear equation, linear range, correlation coefficient, quantitative limit for four aflatoxins
And (3) taking the quantitative limits of 1 time, 2 times and 4 times of each aflatoxin as the blank sample standard adding levels, performing six groups of parallel experiments at each adding level, and detecting according to the extraction and analysis method, wherein the recovery rate results and the relative standard deviation are shown in table 4, and the results show that the standard adding recovery rate of the blank sample is 81.96-93.21%, and the quantitative limits can meet the detection requirements.
Table 4 recovery on standard and relative standard deviation of four aflatoxins (n ═ 6)
The mass concentration of the aflatoxin is taken as an abscissa, the corresponding peak area is taken as an ordinate to draw a standard curve, and according to the standard curve of each aflatoxin and the peak area in a sample, the content of aflatoxin B1 in the embodiment is 15.65 mug/kg, the content of aflatoxin B2 is 5.24 mug/kg, the content of aflatoxin G1 is 4.56 mug/kg, and the content of aflatoxin G2 is 0.57 mug/kg.
Wherein the peak area corresponding to aflatoxin B1 is 468652, the peak area corresponding to aflatoxin B2 is 240081, the peak area corresponding to aflatoxin G1 is 351048, and the peak area corresponding to aflatoxin G2 is 45290.
In the embodiment, the earlier stage extraction treatment is about 30 minutes, the consumed time is short, the operation is simple, the operation is easy to operate, the dosage of the related organic solvent is less, the environmental pollution is less, and compared with the existing immunoaffinity column, the sample purification (Chinese pharmacopoeia) is low in cost, and is more economical and applicable.
TABLE 5 comparison of the parameters of the examples of the invention with those of the prior art
The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, so that all equivalent changes and modifications made according to the scope of the present invention are included in the scope of the claims of the present invention.
Claims (7)
1. A method for extracting and detecting aflatoxin in spina date seeds is characterized by comprising the following steps:
a) crushing the spina date seeds, sieving the crushed spina date seeds by a second sieve to obtain spina date seed powder, placing the spina date seed powder into a centrifuge tube with a plug, and adding methanol or acetonitrile into the centrifuge tube at a material-to-liquid ratio of 1: 1-1: 100; performing vortex oscillation for 1 min-10 min at the rotating speed of 1000 r/min-3000 r/min, and performing ultrasound for 5 min-30 min at the ultrasonic power of 100W-500W; adding 0.1-20 g of anhydrous sodium sulfate or anhydrous magnesium sulfate or sodium chloride, carrying out vortex mixing at the rotating speed of 1000-3000 r/min for 1-10 min, centrifuging at the rotating speed of 1500-4500 r/min for 8-16 min, and taking supernatant;
b) taking supernatant into a centrifuge tube, adding 0.5-10 g of octadecylsilane chemically bonded silica or N-propylethylenediamine for purification, carrying out vortex mixing at a rotating speed of 1000-3000 r/min for 1-10 min, centrifuging at a rotating speed of 1500-4500 r/min for 3-10 min, and filtering through an organic phase filter membrane with a pore size of 0.1-0.6 mu m to obtain a liquid to be detected;
c) after aflatoxin standard use solution is filtered by an organic phase filter membrane with the pore size of 0.1-0.6 mu m, a liquid chromatography-tandem mass spectrometer is adopted to detect the solution to be detected, a chromatographic column is Unitamy C18, a mobile phase A is ammonium formate or ammonium acetate or formic acid, a mobile phase B is methanol, the flow rate is 0.2-0.5 mL/min, the column temperature is 25-40 ℃, the sample injection volume is 1-10 mu L, the ionization mode is electrospray positive ion ionization, and the detection mode is multi-ion reaction monitoring.
2. The method for extracting and detecting aflatoxin in spina date seeds according to claim 1, characterized in that the pore size of the organic phase filtration membrane is 0.2-0.5 μm.
3. The method for extracting and detecting aflatoxin in spina date seeds as claimed in claim 1, wherein the chromatographic column packing is alkylsilane bonded silica gel, the length of the chromatographic column is 100 mm-250 mm, the inner diameter is 2.1 μm-4.6 μm, and the particle size is 1.7 μm-5 μm.
4. The method for extracting and detecting aflatoxin in spina date seeds according to claim 1, characterized in that in the step a), after vortex oscillation is carried out for 3min at the rotating speed of 4000r/min, ultrasonic treatment is carried out for 20min at the ultrasonic power of 500W, after vortex mixing is carried out for 3min at the rotating speed of 4000r/min, centrifugation is carried out for 5min at the rotating speed of 4000 r/min.
5. The method for extracting and detecting aflatoxin in spina date seeds according to claim 1, which is characterized in that N-propylethylenediamine is added for purification in the step b), the mixture is subjected to vortex mixing at a rotating speed of 4000r/min for 3min, and then the mixture is centrifuged at a rotating speed of 4000r/min for 3min and then filtered by an organic phase filter membrane with a pore size of 0.22 mu m.
6. The method for extracting and detecting aflatoxin in spina date seeds according to claim 1, wherein the step of measuring the standard using solution of aflatoxin in the step c) comprises the following steps: taking aflatoxin mixed reference solution with known concentration, diluting with methanol or ethanol or acetonitrile solution with volume fraction of 50-90%, fixing volume, storing at 2-8 ℃ for 1-3 months, and preparing into standard aflatoxin use solution with different concentrations with methanol with volume fraction of 50-90% when in use.
7. The method for extracting and detecting aflatoxin in spina date seeds according to claim 6, wherein the volume fraction of methanol is 70-90%.
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| WO2022262245A1 (en) * | 2021-06-15 | 2022-12-22 | 浙江海正药业股份有限公司 | Method for analyzing and detecting trace aflatoxin in water-soluble fermented medicament |
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Application publication date: 20201229 |